Occurrence:Buckwheat flour is used as a substitute for wheat flour in many recipes. It is frequently used in making savoury pancakes. These are called galettes or crepes de sarasin in France (buckwheat is known as “sarasin” or “blé noir” in France). It is also used in several types of Japanese noodles called soba, ramen, somen, or udon.

Allergy Information:

Buckwheat, despite its name, is not botanically related to wheat. Thus individuals who are allergic to wheat, barley, rye and similar cereals can usually eat buckwheat. Similarly, buckwheat flour can be safely used by individuals with coeliac disease (provided that it has been milled separately from wheat, rye, barley or oats).

Allergy to buckwheat is one of the more common food allergies in Korea and Japan. As with most food allergens, the most common symptoms involve the skin, especially urticaria (hives). Severe symptoms such as anaphylaxis have been reported and also exercise induced anaphylaxis i.e. anaphylaxis which only occurs when consumption is followed within a few hours by exercise such as running.

Buckwheat is not named in the European or American labelling regulations but labelling of buckwheat is mandatory in Japan, reflecting the much higher prevalence of this allergy in Asia.

Yoshimasu et al. (2000) [244] reported 12 patients with the following symptoms: cutaneous symptoms (9/12), wheezing or asthma (6/12), Gastrointestinal (6/12) and one each of discomfort of larynx, shock and anaphylaxis.

Tanaka et al. (2002) [610] reported data from 20 patients with positive CAP to buckwheat of whom 9 were food allergic, one reacted with asthma to buckwheat as an aeroallergen and 10 others did not show immediate symptoms on ingestion. Of the food allergic patients, 2/9 showed cutaneous symptoms only and 7/9 generalized symptoms.

Park et al. (1997) [143] found that 7/12 subjects were RAST positive for BWI-1 but that only one of five sera tested had more than 50% inhibition. The BWI-1 family are candidates for or components of the 9 kDa allergen.

Park et al (2000) [140] transferred proteins onto a 0.45µm nitocellulose membrane by electroblotting. This was blocked with Tris and 5% (w/v) non-fat milk with Tween 20 and incubated with patients' sera at a dilution of 1:10, washed with Tris buffer with Tween 20 and incubated with goat alkaline phosphatase-conjugated anti-human IgE (1:1000), then visualized by reaction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium.

Yoshimasu et al. (2000) [244] transferred proteins onto a nitocellulose membrane by electroblotting. This was blocked with Tris/BSA with Tween 20 and incubated with patients' sera at a dilution of 1:10, washed Tris buffered saline with Tween 20 and incubated with alkaline phosphatase-conjugated anti-human IgE or IgG, which was visualized by reaction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium.

Tanaka et al. (2002) [610] transferred proteins onto a PVDF membrane. This was washed with PBS with Brij 35 and blocked with BSA. The membrane was incubated with patients' sera at a dilution of 1:10, followed by the addition of goat alkaline phosphatase-conjugated anti-human IgE, or IgA (1 :5000) and visualized by reaction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium.

Lee et al. (2001) [396] transferred proteins onto a nitocellulose membrane by electroblotting. This was blocked with Tris/BSA and incubated with patients' sera at a dilution of 1:20, washed Tris buffered saline with Tween 20 and incubated with alkaline phosphatase-conjugated anti-human IgE, which was visualized by reaction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium.

Immunoblotting results:

Matsumoto et al. (2004) [942] report the 24-kDa protein (BW24KD as the most prominent band, which was recognized equally by IgG, IgA, or IgE. The 10 kDa protein (BW10KD) was more strongly IgE reactive comparted with IgG or IgA, in 57% of allergic individuals.

Park et al (2000) [140] identified by immunoblotting 24kD, 19kD, 16kD and 9kD proteins as major allergens. 30 kDa, 43 kDa and 67 kDa allergens were also found. The asymptomatic subjects also reacted to the 24kD, 16kD and 9kD proteins but only one to the 19 kDa proteins (this band is split).

Yoshimasu et al. (2000) [244] found IgE reactive bands between 45-66 kDa and fainter bands at 14 and 18 kDa before reduction. After reduction, 45 kDa, 18 kDa and 14 kDa bands bound IgE strongly. The 6 bands at 45-66 kDa gave a protein band at 24 kDa after reduction which did not bind IgE.

Tanaka et al. (2002) [610] found IgE binding to the 24 kDa protein in 19/20 sera and to the 16 and 19 kDa proteins in 9/10 patients with symptoms. As the 16 kDa protein was resistant to pepsin, they conclude it is associated with anaphylaxis.

Yoshimasu et al. (2000) [244] report that the main IgE binding proteins ran between 45 and 66 kDa on non-reducing gels. With reducing gels, seven bands from 10-20 kDa were seen. There was no 24 kDa allergen. The strongest IgE binding was at 14 and 18 kDa. The N-terminal sequences of the 14 kDa band was KYEGALKRIEGEGCK. This distantly resembles rice phenylalanine ammonia lyase and rice endosperm protein.

Allergen stability:Process, chemical, enzymatic:Stable to cooking in pancakes and noodles. Tanaka et al. (2002) [610] show that the 16 kDa protein is one of the most pepsin resistant buckwheat proteins.

Nature of main cross-reacting proteins:IgE from buckwheat allergic individuals has been reported to cross-reacts with proteins from latex and rice. The 16-19 kDa allergens seem to be related to alpha-amylase/trypsin inhibitor family (including 2S albumins).

Yoshimasu et al. (2000) [244] report that the main IgE binding proteins ran between 45 and 66 kDa on non-reducing gels. With reducing gels, seven bands from 10-20 kDa were seen. There was no 24 kDa allergen. The strongest IgE binding was at 14 and 18 kDa. Park et al. reported the N-terminal sequences of the 16 kDa allergen as RDEGFDLGETQMSSKCMRQVKMNEP, which was reported as distantly homologous to the alpha-amylase/trypsin inhibitors of millet. Yoshimasu et al. report the 18 kDa allergen sequence as RDEGFDLGETQMSSKCMR. This resembles rice dehydrin (residues 84-101) and chilling-inducible protein. As the N-terminal sequences are the same, the 16 and 18 kDa allergens are probably identical. Both are related to the BW8 kDa allergen protein

Nature of main cross-reacting proteins:IgE from buckwheat allergic individuals has been reported to cross-reacts with proteins from latex and rice. The 16-19 kDa allergens seem to be related to alpha-amylase/trypsin inhibitor family (including 2S albumins).

Allergen properties & biological function:Possibly a seed storage protein as it may be homologous to rice alpha-globulin.

Allergen stability:Process, chemical, enzymatic:Most 2S albumins are relatively stable to both heat and proteolysis, probably because the structure consists of alpha-helices, which can quickly refold, crosslinked by 4 disulphide bridges.

Nature of main cross-reacting proteins:

IgE in allergic sera to buckwheat cross-react with those from latex (De Maat-Bleeker & Stapel, 1998) [39] and rice (Yamada et al, 1995) [236]. There are no close homologues in Swissprot with Ber e 1 closest at 29% identity. There is a relationship between this sequence (Nagata et al. 2001 [124]) and that given by Park et al. (2000) for the 16 kDa allergen as shown below

Buckwheat was extracted twice with 0.125 M NaHCO3 at 4°C for 1 h, centrifuged and the supernatants dialyzed against distilled water. The extracts were loaded on an anion-exchange chromatographer (DEAE-TOYOPEARL 650M, Tosoh, Tokyo) that had been equilibrated with Na2HPO4 (20 mM, pH 7). After washing the column with the same buffer, the proteins were eluted with a linear gradient of NaCl ranging from 0 to 1 M NaCl in Na2HPO4 (20 mM, pH 7). The fractions containg the partially purified BW10kD were identified by SDS-PAGE.

Other biochemical information:

This is was initially described as the BW8kD allergen by Nagata et al. (2001) [124] and later called the BW10KD allergen by Matsumoto et al. (2004) [942]. In 1D-immunoblots it may overlap the 9 kDa allergen of Park et al. (1997) [143] and Park et al (2000) [140].

Allergen stability:Process, chemical, enzymatic:Some buckwheat allergens are known to be stable to cooking.

Nature of main cross-reacting proteins:The buckwheat legumins listed above share more than 80% identity with each other and would be expected to cross-react. They would also be expected to cross-react with 11S seed storage globulins from other species of buckwheat such as Q9M642 (fragment) from Fagopyrum gracilipes and Q8LGR7 from Fagopyrum tataricum (Tartarian buckwheat).

Allergen stability:Process, chemical, enzymatic:The inhibitors function after heating, exposure to organic solvents and low pH during extractions. Thus they are either very stable or else can easily refold. They would be expected to survive cooking.

Nature of main cross-reacting proteins:These inhibitors are similar to inhibitors from Amaranth (eg. P80211). IgE in allergic sera to buckwheat cross-react with those from latex (De Maat-Bleeker & Stapel 1998 [39]; Schiffner et al., 2001 [502])

Allergen properties & biological function:These allergens belong to the family of type I potato serine protease inhibitors. These are induced by wounding and are part of the PR-6 family of plant defence proteins. Because rubber plants are wounded in obtaining latex, these are possible, but not established, latex allergens

Allergen purification:Bolozersky et al (1995) [23] followed an initial extraction from the seed by either a trypsin-Sepharose column or Sephadex G-75 followed by chromatography on Mono Q HR 5/5 in 20 mM K, Na-phosphate, pH 6.8. Protein was eluted by a 0-100 mM NaCl gradient. BWI-1 was then purified by reverse-phase HPLC on a aquapore RP-300 column using a acetonitryl gradient. Pandya et al. (1996) [139] stirred 250 g batches of flour with 2.5l of 2% (w/v) NaCl for 90 min. This was filtered and centrifuged. The supernatant was heated to 80 deg. C for 10 min. Ammonium sulphate was added to 60% saturation. The ppt. was disolved in water, dialysed and lyophilized. The protein was disolved in 6M urea in 0.1M acetic acid and fractioned by gel filtration (Sephadex G-50 equilibrated in 0.1M acetic acid). Fractions were lyophilized and applied to an inactivated trypsin column at pH 8.1. The inhibitors were eluted at pH 2.1 in 0.1M KCl, desalted and lyophilized. The protein was then chromatographed on DEAE-Sepharose CL-6B equilibrated with 50 mM Tris, pH 8.4, with a 0-0.3M NaCl gradient. Peaks were rerun on a Synchropak AX300 HPLC column in 10 mM Na-Pi buffer and eluted using gradients 0-0.2M and 0,2-0.6M NaCl. Park et al. (1997) [143] extracted defatted flour with 10 mM sodium phosphate, pH 7.5, containing 0.15M NaCl with agitation for 2 hours at 4°C. After centrifigation (20 minutes at 13000g), ammonium sulfate was added to 100% saturation and precipitate was collected after 24 hours by centrifugation. After dialysis against 10 mM sodium phosphate, pH 7.5, and gel filtration on Sephadex G-50, the trypsin inhibitory fractions were collected. These were further purified and separated on a DEAE-cellulose column equilibrated with 10 mM Tris/HCl pH 8.0, with elution by a linear 0-0.3M NaCl gradient, followed by purification on an FPLC Mono-Q column and RP-HPLC on a Wakosil C-18 column.

Allergen stability:Process, chemical, enzymatic:The inhibitors function after heating, organic solvents and low pH during extractions. Thus they are either very stable or else can easily refold. They would be expected to survive cooking.

Allergen purification:Pandya et al. (1996) [139] stirred 250 g batches of flour with 2.5l of 2% (w/v) NaCl for 90 min. This was filtered and centrifuged. The supernatant was heated to 80 deg. C for 10 min. Ammonium sulphate was added to 60% saturation. The ppt. was disolved in water, dialysed and lyophilized. The protein was disolved in 6M urea in 0.1M acetic acid and fractioned by gel filtration (Sephadex G-50 equilibrated in 0.1M acetic acid). Fractions were lyophilized and applied to an inactivated trypsin column at pH 8.1. The inhibitors were eluted at pH 2.1 in 0.1M KCl, desalted and lyophilized. The protein was then chromatographed on DEAE-Sepharose CL-6B equilibrated with 50 mM Tris, pH 8.4, with a 0-0.3M NaCl gradient. Peaks were rerun on a Synchropak AX300 HPLC column in 10 mM Na-Pi buffer and eluted using gradients 0-0.2M and 0,2-0.6M NaCl.Bolozersky et al (1995) [23] made an extract from seeds and used either a trypsin-Sepharose column or Sephadex G-75 followed in each case by chromatography on Mono Q HR 5/5 in 20 mM K, Na-phosphate, pH 6.8. Protein was eluted by a 0-100 mM NaCl gradient. BWI-1 was then purified by reverse-phase HPLC on a aquapore RP-300 column using acetonitryl and trifluoracetic acid. Park et al. (1997) [143] extracted defatted flour with 10 mM sodium phosphate, pH 7.5, containing 0.15M NaCl with agitation for 2 hours at 4°C. After centrifigation (20 minutes at 13000g), ammonium sulfate was added to 100% saturation and precipitate was collected after 24 hours by centrifugation. After dialysis against 10 mM sodium phosphate, pH 7.5, and gel filtration on Sephadex G-50, the trypsin inhibitory fractions were collected. These were further purified and separated on a DEAE-cellulose column equilibrated with 10 mM Tris/HCl pH 8.0, with elution by a linear 0-0.3M NaCl gradient, followed by purification on an FPLC Mono-Q column and RP-HPLC on a Wakosil C-18 column.

Other biochemical information:A range of allergens have been identified by immunoblotting including ones with Mrs of 24kD, 19kD, 16kD and 9kD proteins.

Park et al. (1997) [143] tentatively identify these sequences as distant homologues of the cysteine rich N-terminal domain found in some vicilins. Alignment with Jug r 2 and AMP2 from Macadamia nut show 13/37 identities including four cysteine conserved in several vicilins. AMP2 has been shown to be cleaved to produce a family of antimicrobial peptides ( Marcus et al. 1999 [1002]).